Materials and Methods for Isolating Phosphopeptides

ABSTRACT

Protein phosphorylation is a major post-translational modification and it plays a pivotal role in numerous cellular functions. We present a composition that includes a soluble nanopolymer core functionalized with groups having an affinity for either metal ion or metal oxides which can be used for phosphopeptide enrichment. Exemplary compounds including PolyMAC-Zr, PolyMAC-Fe and PolyMAC-Ti demonstrate outstanding reproducibility, exceptional sensitivity, fast chelation time, and high phosphopeptide recovery from standard mixtures that include phosphorylated peptides. The composition can be used for phosphoproteome isolation from samples of medicinal, diagnostic or biological interest such as malignant breast cancer cells. Such compositions were used for the quantitative analysis of the changes in the tyrosine phosphoproteome in highly invasive breast cancer cells after induction of Syk kinase, a potent suppressor of tumor growth and metastasis. The composition and method disclosed herein offers an efficient and widely applicable tool for phosphoproteomics.

PRIORITY CLAIM

This application claims the benefit of U.S. provisional patentapplication No. 61/103,268 filed on Oct. 7, 2008 which is incorporatedherein by reference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with support from the government under grantnumber CHE-0645020 awarded by the National Science Foundation thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

This disclosure is directed to materials and methods of purifyingphosphorylated peptides in a heterogenous environment, and their use,for example, in identifying differentially phosphorylated peptides.

BACKGROUND

Reversible protein phosphorylation has been shown to be among the mostwidespread of all known post-translational modifications; for example,it is estimated that about 30% of all human proteins are phosphorylatedat one time or another. This post-translational modification plays animportant role in the regulation of many cellular functions, includinggrowth, differentiation, and signaling. Changes in phosphorylationdynamics within the cell has been linked to the onset and development ofnumerous diseases, for example some forms of cancer. For additionalinformation about the role of protein phosphorylation please seepublication such as: “The Origins of Protein Phosphorylation,” PhilipCohen, Nature Cell Biology 4, E127-E-130 (2002); “ProteinPhosphorylation: A Practical Approach”, Oxford University Press, USA,Dec. 15, (1999); and “Protein Phosphorylation Methods in Enzymology,”Academic Press, May 1, (1998).

Accordingly, in order to understand normal development and metabolism,as well as diseases and various disorders it is crucial to develop abetter understanding of protein phosphorylation. Given its central rolein human, animal and event plant health research related to proteinphosphorylation is of great interest to the scientific community,creating a need for new materials and methods to track phosphorylation,some aspects of the invention described herein address this need.

SUMMARY

Some embodiment include a composition suitable for the recovery ofphosphopeptides from a heterogeneous or homogenous mixture, comprising:a synthetic soluble nanopolymer; at least one phosphonate group; and atleast one metal or metal oxide, the metal or metal oxide having anaffinity for at least one phosphorylated residue in a phosphopeptide,wherein the at least one phosphonate group is attached to said solublenanopolymer and the at least one phosphonate group chelates with the atleast one metal or metal oxide.

In some embodiments the composition further includes: at least onesupport structure; and at least one reactive group, wherein the reactivegroup is attached to the synthetic soluble nanoparticle and the reactivegroup interacts with the support structure. In some embodiments themetal in the composition is selected from the group consisting of: iron,copper, gallium, cobalt, nickel, calcium, zinc, cadmium, silver,palladium, platinum, and ruthenium. In still other embodiments the metalis selected from the group consisting of: titanium, zirconium, aluminum,vanadium, lead, manganese and tin. And in some embodiments thecomposition comprises a metal oxide that is selected from the groupconsisting of: titanium oxide, zirconium oxide and tin oxide, aluminumoxide, vanadium oxide, lead oxide and manganese oxide.

In some embodiments there is at least one reactive group attached to thesynthetic nanopolymer the reactive group may be part of a bi-conjugationcoupling pair, selected from group consisting of: hydrazine-aldehyde,azide-alkyne, thiol-iodoaceto, thiol-maleimide and NHS-amine. In someembodiments the synthetic soluble nanopolymer is selected from the groupconsisting of: polyamidoamine dendrimers, polyallyric amines,polylysine, polyarginine, polyethylene glycol derivatives, and dextranderivatives. And in some embodiment the composition includes a s supportstructure that may include an aldehyde on the surface of the structurethat react with a reactive group on the surface of the solublenanopolymer.

In some embodiments the composition the support structure in thecomposition according is a bead.

While in some embodiments the support structure is selected from thegroup consisting of: columns, films and membranes.

Still another embodiment is a method of enriching for, extracting,purifying or identifying at least one phosphopeptide in a heterogeneousor homogenous mixture, comprising steps of: providing a composition thatinteracts with a composition for the recovery of phosphopeptides, saidcomposition including: a synthetic nanopolymer; a metal or a metaloxide, wherein said metal or metal oxide has an affinity for at leastone phosphorylated residue in a phosphopeptide; a first functional groupthat chelates with the metal or the metal oxide; a solid support; and asecond functional group that conjugates to the solid support, whereinthe first functional group and the second functional groups are attachedto the soluble synthetic nanopolymer; obtaining a sample wherein thesample includes at least one phosphopeptide; and contacting the samplewith the composition.

In some embodiments the method of enriching, extracting, purifying oridentifying a phosphopeptide in a sample further includes the steps of:recovering at least a portion of said composition that was in contactwith the sample; eluting the phosphopeptide from the portion of thecomposition and saving at least a portion of the eluent; and analyzingthe eluent for the presence of at least one phosphopeptide. In someembodiments the method further includes the steps of extracting at leastone phosphopeptide from a sample; and identifying at least onephosphopeptide or the lack thereof which from the sample. In someembodiments the eluent is analyzed by mass spectrometry. While in stillother embodiments the eluent is analyzed by contacting at least aportion of the eluent with at least one antibody that is known to bindto at least one phosphopeptide.

In some embodiment the compositions used in the method for enriching,extracting, purifying or identifying phosphopeptides includes a solublesynthetic nanopolymer selected from the group consisting of:polyamidoamine dendrimers, polyallyric amines, polylysine, polyarginine,polyethylene glycol derivatives, and dextran derivatives. In someembodiments the composition includes a metal is selected from the groupconsisting of: iron, copper, gallium, cobalt, nickel, calcium, zinc,cadmium, silver, palladium, platinum, and ruthenium. In still otherembodiments the metal in the composition is selected from the groupconsisting of: titanium, zirconium, aluminum, vanadium, lead, manganeseand tin. And in some embodiments the composition includes a metal oxideis selected from the group consisting of: titanium oxide, zirconiumoxide and tin oxide, aluminum oxide, vanadium oxide, lead oxide andmanganese oxide.

In some embodiments the composition used to extract a phosphopeptidefrom a mixture or enrich a sample in the same includes a secondfunctional group attached to the synthetic nanoparticle that is part ofa bi-conjugation coupling pair, selected from group consisting of:hydrazine-aldehyde, azide-alkyne, thiol-iodoaceto, thiol-maleimide andNHS-amine. And in some embodiments the composition includes a supportstructure that has an aldehyde on the surface of the support structurethat interacts with the synthetic nanopolymer. In some embodiments thesupport structure is a bead, while in other embodiments the supportstructure is selected from the group consisting of: columns, films andmembranes.

Some embodiments include a kit suitable for identifying phosphorylatedpeptides or the lack thereof, in a given sample in which the kitincludes at least one composition comprising: a synthetic nanopolymer,at least one phosphonate group; and at least one metal or metal oxide,the metal or metal oxide having an affinity for at least onephosphorylated residue in a phosphopeptide, wherein the at least onephosphonate group is attached to said synthetic nanopolymer and the atleast one phosphonate group chelates with the at least one metal ormetal oxide. In some kits the synthetic nanopolymer is selected from thegroup consisting of: polyamidoamine dendrimers, polyallyric amines,polylysine, polyarginine, polyethylene glycol derivatives, and dextranderivatives. In some embodiments the synthetic nanopolymer is a solublesynthetic nanopolymer.

Some embodiments such as a polymer-based metal ion or metal oxidecapturing (PolyMAC) are suitable for the recovery of phosphopeptidesfrom mixtures. The composition, may for example, comprise a solublesynthetic nanopolymer, that includes a first functional group chelatingwith a metal that exhibits affinity for at least one phosphorylatedresidue in a phosphopeptide. In some embodiments the composition has aphosphonate group as the first function group chelating with the metal.In some embodiments, the metal may be selected from the group of metalsincluding Ti, Zr, and Sn and the like, this especially useful if thepolymer is used to isolate singly-phosphorylated peptides. In stillother embodiments, the metal may be selected from the group including Feand Ga this is especially useful if the polymer is used to isolatemulti-phosphorylated peptides. In additional embodiments, the metal maybe selected from the group of metal that includes Co, Ni and the likethis is especially useful if the polymer is used to isolate His-taggedproteins. The polymer may also include a second function group thatconjugates to a solid support. In one embodiment, the second functionalgroup is hydroxylamine, and the solid support is comprised of oxidizedaldehyde beads. The nanopolymer may be selected from the groupconsisting of polyamidoamine dendrimers, polyallyric amines,polylysines, polyarganines, derivatives of PEG and the like.

Another group of embodiments include methods of isolatingphosphopeptides. In some embodiments the methods comprise the steps of:(a) providing a comporoung comprising a soluble synthetic nanopolymer,wherein the nanopolymer includes a first functional group that chelateswith at least one metal that exhibits affinity for at least onephosphorylated residue in a phosphopeptide, and a second functionalgroup that conjugates to a solid support; (b) contacting the nanopolymerwith at least one phosphopeptide in the presence of the at least onemetal. In some embodiments the reagent further includes a solid support,wherein the solid support conjugates with the second functional group inthe nanopolymer. In some embodiments the first functional groupchelating with the metal is a phosphonate group. And the metal may be atleast one metal selected from the group of metals including Ti, Zr, Snand the like this is especially useful if the polymer is used, forexample to isolate singly-phosphorylated peptides; or from another groupincluding Fe and Ga this is especially useful if the polymer is used,for example, to isolate multi-phosphorylated peptides; or from yetanother group including Co and Ni this is especially useful if thepolymer is used, for example, to isolate His-tagged proteins. In someembodiments the second functional group that conjugates to the solidsupport may be hydroxylamine, the solid support may be comprised ofbeads that are attached to an aldehyde, and the synthetic nanopolymermay be a polyamidoamine dendrimer, a polyallyric amine, a polylysine, apolyarginine, a derivative of PEG, a derivative of dextran or the like.

Yet another set of embodiments includes methods for identifyingphosphorylated peptides or the lack thereof which may be associated withcertain diseases, for example, cancer cells. The method comprises stepsof: (a) providing a composition comprising a soluble syntheticnanopolymer, wherein the nanopolymer includes a first functional groupthat chelates with a metal that exhibits affinity for at least onephosphorylated residue in a phosphopeptide, and a second functionalgroup that conjugates to a solid support; (b) contacting the nanopolymerto either a diseased cell lysate or a normal cell in the presence of themetal, and the solid support; (c) extracting at least onephosphopeptides from each lysate; and (d) identifying any phosphopeptideor the lack thereof which is associated with the disease. In oneembodiment the first function group chelating with the metal is aphosphonate group. The metal may be any metal selected from a groupcomprising Ti, Zr, and Sn if the polymer is used to isolatesingly-phosphorylated peptides; or from the group comprising Fe and Gaif the polymer is used to isolate multi-phosphorylated peptides; or fromthe group comprising Co and Ni if the polymer is used to isolateHis-tagged proteins. In one embodiment, the second functional group ishydroxylamine, and the solid support is comprised of aldehyde beads. Thenanopolymer may be polyamidoamine, polyallic amines, or polylysines.

Still another embodiment is a kit for identifying phosphorylatedpeptides or the lack thereof that are associated with certainconditions, including for example the presence of cells in a givensample. The kit includes a compound comprising a soluble syntheticnanopolymer, which includes a first functional group that chelates witha metal that exhibits affinity for at least one phosphorylated residuein a phosphopeptide; a second functional group that binds to a solidsupport; and a solid support. The nanopolymer may be selected from groupconsisting of polyamidoamine dendrimers, polyallyric amines,polylysines, polyarganines, derivatives of dextran, derivatives of PEGand the like. The synthetic nanopolymer may be soluble.

These and other features, aspects and advantages of the disclosure willbecome better understood with reference to the following drawings,description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A diagram illustrated one exemplary synthesis of novel solublephospho-enrichment reagent.

FIG. 2. A chart illustrating a kinetic comparison of differentphosphor-peptide enrichment methods.

FIG. 3. A chart illustrating the relative efficiencies of PolyMAC andbeads coated with TiO₂ nanoparticles used to enrich a sample inphosphorylated peptides.

FIG. 4. Mass spectrometry data showing the relative efficiencies of 2different methods of enriching samples in at least one phosphorylatedpeptide in complex samples using PolyMAC-Ti and TiO₂ beads.

FIG. 5. Mass spectrometry data illustrating the detectable levels of a(1126 Da) phosphopeptide measured in the presence of a large amount ofbeta-lactoglobulin. The spectra in the upper panel were measured beforethe sample was treated with PolyMAC-Ti.

FIG. 6. Tracing showing the amount of a polypeptide in various samplesdrawn at various stages of an enrichment protocol using PolyMAC-Fereagent to capture and later release a phosphopeptide.

FIG. 7. Experimental flowchart illustrating the steps in analyzingbreast cancer cells for phosphorylated polypeptides enriched in suchsample using PolyMAC-Ti.

FIG. 8. Experimental flowchart illustrating the steps in analyzingbreast cancer cells for phosphorylated polypeptides enriched in suchsample using PolyMAC-Ti, this protocol includes the step of subjectingthe samples to an anti-phosphotyrosine antibody affinity purification(pYIP) step before adding PolyMAC-Ti.

FIG. 9. Schematic diagram illustrating the components of the Tet−operator plasmids that were used to control the expression of Syk kinasein invasive breast cancer cells.

FIG. 10. Gels showing the amounts of Syk in Tet− and Tet+ MDA-MB-231breast cancer cells (upper gel) and Syk and GAPDH (lower image)expression with different amount of Tet treatment.

FIG. 11. Bar graph illustrating the comparison of the relativeselectivity of PolyMAC-Ti and TiO₂ nanoparticles to engagephosphopeptides in a given sample. In the samples treated withPolyMAC-Ti about 95% of the peptides identifies from the Tet+/MDA-MB-231cells were found to be tyrosine phosphorylated peptides. In the samplestreated with TiO₂ about 76% of the peptides identifies from theTet+/MDA-MB-231 cells were found to be tyrosine phosphorylated peptides.

FIG. 12. Selected phosphopeptides present in Syk-induced samples, “p”adjacent to a residue indicates that the residue may be phosphorylated.

FIG. 13. A bar graph illustrating some of the tyrosine phosphorylatedpeptides that were identified in breast cancer cells using PolyMAC-Ti.

DESCRIPTION

While the concept of the present disclosure are illustrated anddescribed in detail in the drawings and the description herein, such anillustration and description are to be considered as exemplary and notrestrictive in character, it being understood that only the illustrativeembodiments are shown and described and that all changes andmodifications that come within the spirit of the disclosure are desiredto be protected.

Protein phosphorylation and de-phosphorylation play a fundamental rolein the both normal cell development and in a myriad of diseases anddisorders. For example, the phosphorylation state of various proteinsplays a central role in process such as apoptosis, aggressive growth,abnormal proliferation, the invasive growth of tumor cells, metastasisof cancer cells as well as normal cell growth, statis and death.Accordingly, there is a glaring need to identify proteins that arephosphorylated in both normal and abnormal cells as well as trackingtheir phosphorylation states in various stages of cell growth,development and pathology.

To that end, numerous methods have been adapted for analyzingphosphorylation states one of the most commonly used methods includeslabeling the proteins with ³²P. Despite its sensitivity and robustness,this technique is not suitable for global phosphoproteomic studies andbiomarker discovery. Recently, mass spectrometry has emerges as themethod of choice for studying in phosphorylation and the like, thisadvance in the art has contributed greatly to making discovery-basedphosphoproteomic analysis a reality. Unfortunately, phosphorylatedproteins often exist in substoichiometric ratios when compared tounmodified proteins, making it difficult to identify phosphorylatedpolypeptide, in particular it is difficult to use this technique toidentify the exact site of polypeptide phoshorylation. In order toovercome this limitation, several phosphopeptide enrichment techniqueshave been developed, which enable researcher to more confidentlyidentify phosphorylated polypeptides. Major methods that have beendeveloped include, for example, the following: a method that includesantibody affinity-based binding, a method that involves the chemicalderivatization of phospho-sites, and a method that uses a metal ionaffinity-based assay.

The antibody affinity-based methods have been widely used for theselective isolation of phosphopeptides, unfortunately the highspecificity and low efficiency of this technique has made it lessdesirable for specific uses such as screening for unknown phosphorylatedproteins. Currently, the technique is most widely used only for theidentification of phosphorylated proteins that interact withphosphotyrosine-directed antibodies.

The chemical modification approach has also been used frequently perhapsthe most notable version of this method tracks the β-elimination ofphosphates from phosphorylated serine/threonine sites and a subsequentMichael addition of a label to the site. The usefulness of thistechnique lies in the chemical substitution of the MS-labile phosphategroup, allowing for a more confident identification of the modifiedresidues. Additionally, attaching certain labeling groups to thepolypeptide during the Michael addition step provides an opportunity forthe affinity purification of the labeled polypeptide. Nonetheless, thismethod suffers from a number of drawbacks, including poorreproducibility and side reactions, making identification morechallenging.

One of the most frequently utilized methods for the isolation ofphosphopeptide is IMAC (immobilized metal affinity chromatography). Theapproach is based on the selective affinity of a metal ion for thephosphate group these methods usually use metals such as Fe (III) and Ga(III). However, this method suffers from poor selectivity, perhaps dueto some affinity of Fe+3 and Ga+3 for acidic residues, its lowspecificity makes it a less attractive method than it would beotherwise. In order to overcome this pitfall, some variations of thetechnique include modifying acidic residues by methyl esterificationbefore running the assay this approach has used with some successdecrease nonspecific binding thereby increasing the utility of themethod. Unfortunately, this modification step may be problematic due tooften incomplete esterification and side reaction products thatcomplicate the mass spectrometry analysis of the phosphorylatedpolypeptide.

A strategy for isolating phosphorylated polypeptides uses an oxideversion of a metal. Especially successful metal oxides put to this useinclude TiO₂, ZrO₂ and Al(OH)₃ which may be used in conjunction withbeads, chromatography columns and the like for the isolation ofphosphopeptides. Certain metal dioxides, for example, TiO₂ and ZrO₂,have demonstrated an exceptionally high selectivity for and strongaffinity for the chelation of phosphorylated residues, providing anadvance in the field of phosphopeptide isolation. Despite the manyadvantages of titania- and zirconia-functionalized beads, there are somenegative aspects of this approach including, for example, including slowreaction kinetics, modest phosphopeptide recovery, and problems withreproducibility. Potentially, at least some of these problems may be dueto heterogeneous reaction conditions caused by the use of solid-phasebeads and nanoparticles. Heterogeneous reaction conditions may lead toan unequal distribution of the reactive groups, reduced access tofunctional groups, nonlinear kinetic behavior, and solvation problems.Accordingly, there is still room for improvement even when using metaloxides in conjunction with beads to isolate phosphorylated polypeptides.

Some embodiments of the instant invention seek to address some of theselimitation by introducing materials and methods for phosphopeptideenrichment that include, for example, PolyMAC-Zr (a polymer-based metalaffinity capture material that includes zirconia). Compounds such asPolyMAC-Zr and the like unexpectedly overcome the problems surroundingheterogeneous reaction conditions found with simple metal oxide/beadbased systems. Reagents such as PolyMAC-Zr include a solublepolyamidoamine synthetic nanopolymer (e.g. a dendrimer) that includes ahyper-branched surface which can be functionalized with various chemicalgroups. In addition to increased solubility, the advantages of usingdendrimers include their high structural and chemical homogeneity,compact spherical shape, and readily controllable surfacefunctionalities. Many of these compounds also possess a unique abilityto cross cell membranes, and have been widely used to deliver vaccines,drugs, and genomic materials into cells. Accordingly, dendrimers can befunctionalized with materials such as zirconia to create materials thatare well suited for the robust and efficient isolation ofphosphopeptide. Such reagents have demonstrated reproducibly highselectivity, favorable kinetics and excellent levels of phosphopeptiderecovery. As discussed below, in one exemplary application, some of thismaterial was used to analyze a sample for the presence ofphosphopeptides that are thought to be created as a result of Sykkinase-induced phosphorylation. This is of special interest because theactivity of Syk-kinase is different in highly invasive breast cancercells than it is in non-cancerous breast cells.

Some embodiment include a composition, or method of using the same, thatincludes, but are not limited to different synthetic nanopolymers suchas PAMAM polyaminoamine dendrimer, polyallyricamine, polylysine,polyarginine, PEG (polyehtylene glycol) derivatives, dextranderivatives, and the like.

Some embodiments include at least one metal or metal oxide that isattached to the synthetic nanopolymer and that interacts with at leastone phosphopeptide, phosphoprotein or phospho-polypeptide including, butare not limited to, iron, gallium, copper, cobalt, nickel, cadmium,ruthenium, mercury, gadolinium, aluminum, zinc and the like.

Some embodiment include at least one metal oxide that is attached to thedendrimer and that interacts with at least one phosphopeptide,phosphoprotein or phospho-polypeptide including, but not limited to,titanium oxide, zirconium oxide, vanadium oxide, tin oxide, lead oxideand the like.

In some embodiments the dendrimers may be attached to other groups suchas support structures including, but not limited to, beads, membranes,columns, using various chemically active moieties, for examplebi-conjugation pairs such as hydrazine-aldehyde, azide-alkyne,thiol-iodoaceto, thiol-maleimide, NHS N-hydroxysulfosuccinimide)-amineand the like.

Spleen tyrosine kinase (Syk) is a 72 kDa signaling protein involvedprimarily in B cell antigen receptor signaling. The expression of Syk islower in malignant breast cells than in normal breast cells. Moreover,when Syk was transfected into malignant breast carcinoma cells, it wasfound to act as a tumor suppressor, acting at least in part by,inhibiting cell motility and proliferation. Some of the most dramaticchanges observed by transfecting breast cancer cells with Syk includedreduced malignancy, decreased cancerous cell proliferation, anddecreased metastasis. To the best of our knowledge to date there havebeen no published reports that identify the physiological substrates ofSyk in breast cancer cells. Accordingly, elucidating Syk action in moredetail will increase our understanding of breast cancer onset andprogression, and should aid in the discovery and development oftherapeutic targets to treat breast cancer and similar diseases.

Using PolyMAC-Ti and PolyMAC-Zr we quantitatively analyzed the changesin invasive breast cancer cell phosphoproteome before and after theinduction of Syk kinase expression. As discussed further below proteinsfrom two sets of malignant carcinoma cells (−Syk or +Syk) were digested,isotopically labeled and phosphopeptides isolated using PolyMAC-Zr, thesamples were analyzed using two-dimensional microcapillary LC-tandemmass spectrometry for identification and relative quantification ofphosphorylated proteins in the samples.

Experimental Methods and Results

Referring now to FIG. 1, a diagram illustrating some of the componentand steps in the process of synthesizing a composing according to someembodiments of the invention. Briefly, a support structure, such a beadincludes surface groups, such as aldehydes, that interact with at leastone reactive group (a hydrazine) comprising a dendrimer such as PAMAMdendrimer. A second group on the surface of PAMAM dendrimer includes afunctional group such as phosphonate which can be formed by the reactionof a carboxyethyl-phosphoric acid with amine groups of the dendrimer toform a phosphonate group that chelates either or both metal ions andmetal oxides.

1. An Exemplary Synthesis of PolyMAC-Ti

One protocol suitable for the synthesis of compounds such as PolyMAC-Tiis as follows. Dry 200 μl of PAMAM (polyamidoamine) dendrimer generation4 solution (provided as 10% (wt/vol) in methanol, such material avaiablefrom Sigma-Aldrich is placed in a microfuge tube. Bring-up resolubilizedried dendrimer in 2 ml of anhydrous DMSO (dimethyl sulfoxide) andtransfer into a 10-ml round-bottom flask with a magnetic stir bar. In amicrofuge tube, add 5.5 mg of Boc-amino-oxyacetic acid, 10 mg of HOBt(hydroxybenzotriazole) and 10 μl of DIPCI (1,3-diisopropylcarbodiimide);dissolve in 1 ml of DMSO and incubate for about 30 min at roomtemperature. Add the mixture into the round bottom flask containingdendrimer and stir overnight. Dialyze the solution against water for 7-8hours using, for example, Snakeskin® pleated dialysis tubing (3,500MWCO, 22 mm dry diameter, avaiable from Pierce) to remove any remainingunreacted reagents (replace water periodically during dialysis).Transfer the solution into an Amicon Ultra centrifugal filter device (5kDa MW cutoff; avaiable from Millipore) and concentrate it to a volumeof about 2 ml or less. Bring the mixture up to 2 ml with water andtransfer into a clean 10-ml round-bottom flask with a stir bar. Add 1 mlof 250 mM MES (2-(N-morpholino)ethanesulfonic acid; pH 5.8) (it can beused it to wash the remaining reagent out of the Amicon filter), 16 mgof carboxyethyl-phosphoric acid and 160 mg of EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) into theflask, and stir overnight to functionalize the dendrimer with phosphonicacid. Dialyze the solution against water for 5-6 hours to remove anyunreacted reagents (replace water periodically during dialysis).Concentrate again using Amicon filter tubes to the final volume of 1.5ml. At this point, the mixture should be stored at −20° C. and theremaining synthesis steps can be done three times to using 500 μl of themixture at one time (will produce three batches of reagent). Mix 500 μlof the above solution and 500 μl of 100 mM zirconium oxychloride(TiOCl₂) and incubate for 1 hour with agitation at room temperature tochelate zirconia with phosphonic acid groups on the dendrimer. Dialyzethe solution overnight to remove any unbound zirconia (replace dialysissolution periodically during dialysis). Remove from dialysis and dry thesolution in a glass tube under vacuum using SpeedVac concentrator(Savant). Dissolve the reagent in 100 μl of water and add 400 μl of 100%TFA (trifluoroacetic acid), incubate for 1 hour with agitation at roomtemperature to remove the Boc protecting group. Dry the sample andresolubilize in 750 μl of DMSO. Add 250 μl of the 4:1 mixture of 0.1%HCl in water and DMSO into the reagent solution.

2. An Exemplary Synthesis of Aldehyde-Beads.

Transfer 200 mg of controlled pore glass (CPG) beads into a Bio-spin®disposable chromatography column (Bio-Rad). Dissolve 92 mg ofFmoc-serine-OH, 38 mg of HOBt (hydroxybenzotriazole) and 90 μl of DIPCI(1,3-diisopropylcarbodiimide) in 500 μl of DMF (dimethylformamide). Addthe solution into the column containing beads and rotate the columnend-over-end overnight at room temperature to couple Fmoc-serine groupto the beads. Wash the beads with 4 ml of DMF. Add sequentially 250 μlof DMF, 250 μl of dichloromethane, 300 μl of pyridine, and 200 μl ofacetic anhydride to the beads to block the remaining amines; rotateend-over-end for 1 hour at room temperature. Wash the beads three timeswith 1 ml of dichloromethane and three times with 1 ml of DMF. Addsequentially into the column 800 μl of DMF and 200 μl of piperidine toremove the Fmoc group; rotate end-over-end for 1 hour at roomtemperature. Wash the beads three times with 1 ml of DMF and three timeswith 1 ml of dichloromethane. The resulting serine beads should be driedcompletely using SpeedVac concentrator and stored at 4° C. Oxidation:transfer 5-7 mg of serine beads into a frit-based spin column(Bocascientific) and add 200 μl of oxidation solution (8.5 mg of sodium(meta) periodate in 200 μl of 40 mM acetic acid/sodium acetate solution)and incubate for 30 minutes with agitation in the dark at roomtemperature. The resulting aldehyde beads should be used on the sameday.

3. Capturing Phosphopeptides Using PolyMAC-Ti

Resuspended a peptide mixture in 100 μl of the mixture of 150 mM aceticacid/100 mM sodium acetate buffer in 30% ethanol (pH 4.90). Add 10 μl ofsynthesized metal oxide-functionalized dendrimer solution to thepeptides solution and incubate 1-2 minutes with agitation at roomtemperature to allow chelation of phosphopeptides tozirconia-functionalized nanopolymer. Wash the aldehyde beads (oxidizedfor about 30 minutes before use) with 200 μl of 0.1% TFA in thefrit-based spin column by centrifuging them down at about 7,000 rpm forabout 30 seconds. Add the sample reaction mixture into the spin columncontaining aldehyde beads and incubate for 1 hour with vigorousagitation at room temperature. Remove the sample flow-through bycentrifuging the beads down at about 7,000 rpm for about 30 seconds.Wash the beads by agitating the for about 5 minutes at room temperaturewith: a) 100 μl of loading buffer, b) 100 μl of 1% acetic acid in 80%acetonitrile solution (twice), and c) 100 μl of water by centrifugingthe beads down at about 7,000 rpm for about 30 seconds. Elute the boundphosphopeptides by incubating the beads with 100 μl of 400 mM ammoniumhydroxide (NH₄OH) for 5 minutes (twice) with agitation at roomtemperature. Centrifuge down the beads at about 7,000 rpm for about 30seconds, collect and combine the eluents. Dry the eluents under vacuumand resolubilize in 0.1% formic acid for mass spectrometry analysis.

4. Using PolyMAC to Recover Phosphopeptides

Using PolyMAC-Ti or PolyMAC-Zr based methods to recover phosphpeptidesdemonstrated faster recovery, higher selectively and greater yieldcompared to some other methods used. Referring now to FIG. 2 a kineticcomparison of enrichment methods assays were carried out on similarsamples enriched from phosphopeptides using 3 different methods thesamples were analyzed by mass spectrometry. Briefly, a standard peptidemixture, comprising angiotensin II peptide (m/z 1046) and itsphosphorylated form (m/z 1126 Da), was incubated with IMAC, TiO₂, orPolyMAC-Ti, respectively. At “0 hrs”, a sample was taken from eachreaction mixture before the addition of the enrichment reagents. Afterthe reagents addition, 1 μl sample of each reaction was taken out at theindicated times and analyzed by MALDI TOF/TOF mass spectrometer tocompare the phosphopeptide binding kinetics of the three methods. Asillustrated by the data presented in FIG. 2 PolyMAC capturesphosphopeptides more efficiently than either beads functionalized withTiO₂ or IMAC.

Referring now to FIG. 3 a bar graph illustrating a comparison ofselective phosphopeptide enrichment methods. After incubating a samplethat includes a mixture of phosphopeptides, with either TiO₂ coatedbeads or the inventive composition including PolyMAC-Ti attached to abead, the flow-through was collected and the beads were washed to removenonspecific binding peptides. Next the bound peptides were eluted offthe beads. All of the flow-through, wash, and elution solutions wereanalyzed by LTQ-Orbitrap™ in order to compare the binding selectivity ofthe two phosphopeptide enrichment methods. It appears that PolyMAC-Zridentified almost 200 more phosphopeptides from the same sample than didthe method relying on TiO₂.

Referring now to FIG. 4 a comparison of the yield and extent ofenrichment between two different methods used to enrich inphosphopeptides in complex samples. After the elution of the boundphosphopeptides, the original amount of the unphosphorylated angiotensinII peptide—with a heavy isotope tag (m/z 623.3 for doubly chargedspecies) was added into each elution solution to be used as an internalstandard. The resulting mixture was analyzed by LTQ-Orbitrap to comparethe recovery yield of phosphopeptide enrichment between the two methodstested. Recovery using the PolyMAC-Ti based method is greater than 90%whereas recovery using the TiO₂ method is less than 10%.

Referring now to FIG. 5, enrichment of phosphopeptide (m/z 1126) in abackground including a large amount of beta-lactoglobulin. A, 1:100mixture that included phosphorylated angiotensin II (MW 1126 Da) andunphosphorylated peptides made from β-lactoglobulin protein wereincubated with the compound PolyMAC-Ti. After the enrichment at time “0hrs” the elution solutions were analyzed by MALDI TOF/TOF to examine theefficiency of phosphopeptide enrichment from the high background ofunphosphorylated peptides. Before enrichment (upper panel and inserttherein), the phosphopeptide was almost invisible due to the presence ofa much larger amount of nonphosphopeptides (100 times higher) in thesample. In contrast, after enrichment (lower panel), only thephosphopeptides were found in the elution, evidence of PolyMAC-Zraffinity for the phosphorylated peptides.

5. Another Exemplary Composition PolyMAC-Fe, is Used to Purify aMultiply-Phosphorylated Peptide.

PolyMAC-Fe is another type of exemplary reagent that is suitable forcapturing phosphopeptides. The synthesis of this reagent is similar tothe synthesis of PolyMAC-Ti described in the above, except that thepolymer is activated with Fe (III). The protocol to use PolyMAC-Ti toenrich a sample in a phosphorylated peptide is also similar to howPolyMAC-Ti is used and may include the following steps: (1) Incubate apeptide mixture with PolyMAC-Fe for about 2 min; (2) Addaldhyde-functionalized solid phase at pH 4.5 m and incubate for about 30min; (3) Centrifuge to remove the supernatant; (4) Wash the beads; (5)Elute the sample with base; and (6) Subject the elutant to LC-MSanalysis or some other means of detecting the presence of a polypeptidein the sample.

PolyMAC-Fe preferably binds multiply-phosphorylated peptides, whilePolyMAC-Ti preferably binds to singly-phosphorylated peptides.Accordingly, these 2 reagents are complementary, and the combination ofthe 2 of them covers the majority of phosphopeptides. Referring now toFIG. 6, the peak with a molecular weight of 1046 corresponds toangiotensin II and the ion peak with a molecular weight of 1126corresponds to phosphorylated angiotensin. The spectra were obtainedusing MALDI-TOF/TOF (ABI 4800). (A) At 0 Hr.; (B) after 30 s ofincubation with the capture reagent indicating the phosphopeptide(m/z1126 was completely, or nearly completely, captured by PolyMAC-Fe);(C) The flow-through did not include a detectable level of anyphosphopeptide; (D) The washing step did not cause any loss of adetectable amount of phosphopeptide; (E) Elution from the reagentyielded mainly phosphopeptide (the amount of non-phosphopeptide isestimated to be less than about 5%); (F) Data gathered by analyzing asample that included adding the same amount of 1046 into the elutionsample as was included in the standard these results indicate the yieldof this recovery process is about 40%.

6. Using the Exemplary Reagent PolyMAC-Ti's in the Analysis of BreastCancer Cell Phospho-Proteomics

Referring now to FIG. 7 a flow chart highlighting some of the steps in aprotocol designed to identify phosphorylated polypeptides in breastcancer cells using various methods of enriching a sample in suchproteins. Briefly, breast cancer cells that did not over express thekinase Syk (upper branch) and breast cancer cells that did over-expressthe kinase Syk were obtained. Proteins recovered from these cells weredigested to create a mixture of peptides and the mixture was contactedwith PolyMAC-Ti. After washing, the bound peptides were eluted from thePolyMAC and analyzed by mass spectrometry. The patterns of polypeptidesin the 2 samples were compared.

Some cancers are thought to involve abnormal patterns ofphosphorylation. For example, some forms of breast cancer the singlemost common form of cancer diagnosed in American women are thought toinvolve abnormal phosphorylation states. Spleen Tyrosine Kinase (Syk) isan enzyme that has been shown to missing or at least present atabnormally low levels in some breast cancer cells. For an additionaldiscussion of Syk activity in highly invasive breast cancer cells pleasesee articles such as Turner, et al., in Immunology Today, 21:148 (2000)and Coopman et al., in Nature 406:742 (2000).

Referring now to FIG. 8 the outline of an experimental protocol foranalyzing for the presence of Syk-induced changes in phospho-proteome inbreast cancer cells. Briefly, 2 sets of invasive cancer MDA-MB-231 cells(Syk-negative or Syk-induced) are lysed, the proteins digested withtrypsin, and the resulting peptides are first enriched for tyrosinephosphopeptides. PolyMAC-Ti is used to further isolate tyrosinephosphopeptides and LTQ Orbitrap MS/MS is used to identify and determinethe relative amount of the phosphoproteins in the samples.

Referring now to FIG. 9 a schematic outline of the constructs (andcontrols kinase Syk expression repressed) made and used to transfectMDA-MB-232 breast cancer cells. For additional details on how theseconstructs were created see, for example, “Role of the Protein TyrosineKinase Syk in Regulating Cell-Cell Adhesion and Motility in BreastCancer Cells,” Zhang X. et al., Mol. Cancer Res. 7(5):634-44 (2009).Referring now to FIG. 10, Western Blots of whole cell lysates run usingsample collected from the human breast cancer cell line MDA-MB-232 cellstransfected with inducible Syk expression systems that were eithertreated with Tet (+) or were not treated with Tet (−). The upper panelshows a gel showing only the region of the gel expected to include Syk.The lower gel shows portions of the gel that are expected to show Sykand GAPDH, lanes of this gel moving from left to right were loaded withthe same amount of proteins recovered from cells treated with 0, 2, 5,20, 100 and 1,000 ng mL⁻¹ of Tet in cell culture.

Referring now to FIG. 12, this figure shows tyrosine phosphopeptidesisolated from whole cell extracts of MDA-MB-232 cells that weretransfected with the Syk vectors described in the above. Samples wererun in parallel to enrich them in the phosphopeptides using eitherPolyMAC-Ti or TiO₂. All of the phosphopeptides listed in FIG. 12 wereidentified in Syk induced samples, the boxes highlight some of moreinteresting proteins that were identified in the samples. All of thephosphopeptides included in FIG. 12 appear to be absent in lysates ofcells made from Syk-negative cells.

Referring now to FIG. 13 this figure illustrated the quantitativedifferences between various tyrosine kinases in Syk− and Syk+ celllines. In each pair of bars the left most bar (blue) represents proteinsin MDA-MB-232 cells transfected with Tet−; while the right most bar(red) represents proteins in MDA-MB-232 cells transfected with Tet+.Interestingly, many tyrosine containing peptides, including TyrosineKinase Yes, Stathmin-2 and Tyrosine kinase Fyn, Kinase suppressor ofRas-2 are less phosphorylated in the presence of the Syk than those inthe absence of Syk, indicating that the sub-phosphorylation of someproteins may contribute to the malignancy of the cancer cell.

It should be understood, of course, that the foregoing relates toexemplary embodiments of the invention and that modifications may bemade without departing from the spirit and scope of the invention as setforth in the following claims. It should be understood, of course, thatthe foregoing relates to exemplary embodiments of the invention and thatmodifications may be made without departing from the spirit and scope ofthe invention as set forth in the following claims.

1. A composition for the recovery of phosphopeptides, comprising: asynthetic soluble nanopolymer; at least one phosphonate group; and atleast one metal or metal oxide, the metal or metal oxide having anaffinity for at least one phosphorylated residue in a phosphopeptide,wherein the at least one phosphonate group is attached to said solublenanopolymer and the at least one phosphonate group chelates with the atleast one metal or metal oxide.
 2. The composition according to claim 1,further including: at least one support structure; and at least onereactive group, wherein the reactive group is attached to the syntheticsoluble nanoparticle and the reactive group interacts with the supportstructure.
 3. The composition according to claim 1, wherein the metal isselected from the group consisting of: iron, copper, gallium, cobalt,nickel, calcium, zinc, cadmium, silver, palladium, platinum, andruthenium.
 4. The composition according to claim 1, wherein the metal isselected from the group consisting of: titanium, zirconium, aluminum,vanadium, lead, manganese and tin.
 5. The composition according to claim1, wherein the metal oxide is selected from the group consisting of:titanium oxide, zirconium oxide and tin oxide, aluminum oxide, vanadiumoxide, lead oxide and manganese oxide.
 6. The composition according toclaim 1, wherein the reactive group is a bi-conjugation coupling pair,selected from group consisting of: hydrazine-aldehyde, azide-alkyne,thiol-iodoaceto, thiol-maleimide and NHS-amine.
 7. The compositionaccording to claim 1, wherein the synthetic soluble nanopolymer isselected from the group consisting of: polyamidoamine dendrimers,polyallyric amines, polylysine, polyarginine, polyethylene glycolderivatives, and dextran derivatives.
 8. The composition according toclaim 2, wherein the support structure includes an aldehyde on thesurface of the structure that react with the reactive group on thesoluble nanopolymer.
 9. The composition according to claim 2, whereinthe support structure is a bead.
 10. The composition according to claim2, wherein the support structure is selected from the group consistingof: columns, films and membranes.
 11. A method for enriching for aphosphopeptide, comprising steps of: providing a composition thatinteracts with a composition for the recovery of phosphopeptides, saidcomposition including: a synthetic nanopolymer; a metal or a metaloxide, wherein said metal or metal oxide has an affinity for at leastone phosphorylated residue in a phosphopeptide; a first functional groupthat chelates with the metal or the metal oxide; a solid support; and asecond functional group that conjugates to the solid support, whereinthe first functional group and the second functional groups are attachedto the soluble synthetic nanopolymer; obtaining a sample wherein thesample includes at least one phosphopeptide; and contacting the samplewith the composition.
 12. The method according claim 11, furtherincluding the steps of: recovering at least a portion of saidcomposition that was in contact with the sample; eluting thephosphopeptide from the portion of the composition and saving at least aportion of the eluent; and analyzing the eluent for the presence of atleast one phosphopeptide.
 13. The method according claim 12, wherein theeluent is analyzed by mass spectrometry.
 14. The method according claim12, wherein the eluent is analyzed by contacting at least a portion ofthe eluent with at least one antibody that is known to bind to at leastone phosphopeptide.
 15. The method according to claim 11, wherein thesoluble synthetic nanopolymer is selected from the group consisting of:polyamidoamine dendrimers, polyallyric amines, polylysine, polyarginine,polyethylene glycol derivatives, and dextran derivatives polyamidoaminedendrimer.
 16. The method according to claim 11, wherein the metal isselected from the group consisting of: iron, copper, gallium, cobalt,nickel, calcium, zinc, cadmium, silver, palladium, platinum, andruthenium.
 17. The method according to claim 11, wherein the metal isselected from the group consisting of: titanium, zirconium, aluminum,vanadium, lead, manganese and tin.
 18. The method according to claim 11,wherein the metal oxide is selected from the group consisting of:titanium oxide, zirconium oxide and tin oxide, aluminum oxide, vanadiumoxide, lead oxide and manganese oxide.
 19. The method according to claim11, wherein the second functional group is part of a bi-conjugationcoupling pair, selected from group consisting of: hydrazine-aldehyde,azide-alkyne, thiol-iodoaceto, thiol-maleimide and NHS-amine.
 20. Themethod according to claim 11, wherein the support structure includes analdehyde on the surface of the structure.
 21. The method according toclaim 11, wherein the support structure is a bead.
 22. The methodaccording to claim 11, wherein the support structure is selected fromthe group consisting of: columns, films and membranes. extracting atleast one phosphopeptides from each lysate; and identifying at least onephosphopeptide or the lack thereof which from the sample.
 23. A kit foridentifying phosphorylated peptides or the lack thereof, comprising: asynthetic nanopolymer, at least one phosphonate group; and at least onemetal or metal oxide, the metal or metal oxide having an affinity for atleast one phosphorylated residue in a phosphopeptide, wherein the atleast one phosphonate group is attached to said polyamidoamine dendrimerand the at least one phosphonate group chelates with the at least onemetal or metal oxide.
 24. The kit according to claim 23, wherein thesynthetic nanopolymer is selected from the group consisting of:polyamidoamine dendrimers, polyallyric amines, polylysine, polyarginine,polyethylene glycol derivatives, and dextran derivatives.